Bridging Integrator-2(Bin2) nucleic acid molecules and proteins and uses therefor

ABSTRACT

The present invention provides Bin2 sequences and proteins encoded thereby. Also provided are compositions and methods utilizing these sequences and proteins in the diagnosis and treatment of blood disorders, including hepatocarcinoma. Further provided are oligonucleotides derived from sequences encoding Bin2, as well as compositions and methods utilizing same for diagnostic and therapeutic purposes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This is a 371 of PCT/US/00/23723, filed Aug. 30, 2000, which claims thebenefit of the priority of U.S. Patent Application No. 60/151,554, filedAug. 31, 1999, now abandoned.

This work was supported in part by Grant Nos. DAMD17-96-1-6324 and DAMD17-98-1-8508 from the US Army Breast and Prostate Cancer ResearchPrograms. The US government has certain rights in this invention.

FIELD OF THE INVENTION

This invention relates generally to cancer diagnosis and therapy, andmore specifically, to cancers associated with over- or underexpressionof Bin1 or other members of the BAR family of adaptor proteins.

BACKGROUND OF THE INVENTION

Bin1/Amphiphysin/RVS (BAR) proteins are a family of adaptor proteinsimplicated in a diverse set of cellular processes, includingtumnorigenesis, cell survival, differentiation, and nerve synapticactivity. BAR proteins share a common N-terminal BAR domain also termedthe RVS domain. While BAR proteins share a common domain (BAR), theyappear to have divergent physiological functions. As one example,amphiphysin is a neuronal protein of this family which is implicated insynaptic vesicle endocytosis [Wigge and McMahon, Trends Neurosci. 21:339-344 (1998)]. Amphiphysin is also a paraneoplastic autoimmune antigenin cancers of the breast, lung, and other tissues [Antoine et al, Arch.Neurol. 56: 172-177 (1999); Dropcho, Ann. Neurol. 39: 659-667(1996);Folli et al., N. Engl. J. Med. 328: 546-51 (1993)].

Bin1 (Bridging INtegrator-1) is a second, ubiquitous BAR protein thatwas initially identified in mammalian cells through its ability tointeract with and inhibit the oncogenic properties of c-Myc [Sakarnuroet al., Nature Genet. 14: 69-77 (1996)]. Ubiquitous Bin1 isoforms thatlocalize to the nucleus have tumor suppressor properties and have beenimplicated in growth control, differentiation, and programmed cell death[Mao et al., Genomics 56: 51-58 (1999); Prendergast, Oncogene 18:2966-2986 (1999); Sakamuro et al. 1996, cited above; Wechsler-Reya etal., Mol. Cell. Biol. 18: 566-575 (1998)].

Other members of the BAR family include the yeast proteins RVS 167 andRVS161, which are believed to have some negative role in cell growthregulation. There exists a need in the art for compositions and methodsuseful for diagnosis and treatment of conditions characterized byinappropriate cell growth control, or disorders affecting cell survival,differentiation, endocytosis, and actin organization.

SUMMARY OF THE INVENTION

The present invention provides a novel member of theBin1/Amphiphysin/RVS (BAR) proteins, termed herein Bin2. Bin2 proteins,nucleic acids, and other Bin2 compositions of the invention have avariety of uses related to regulation of cell growth control, cellsurvival, differentiation, endocytosis and actin organization, as wellas for the diagnosis and treatment of conditions associated withaberrant cell behavior.

In one aspect, the present invention provides a Bin2 protein. In onedesirable embodiment, the protein has the 564 amino acid sequence of SEQID NO:2. In another embodiment, the present invention provides a Bin2peptide or protein selected from the group consisting of a fragment ofBin2 comprising at least 8 amino acids in length. In one embodiment afragment of this invention is at least 8 contiguous amino acids inlength and is selected from amino acids 1 to 13 of SEQ ID NO:2. Inanother embodiment, a fragment of this invention is at least 14 aminoacids in length and includes amino acids 23 to 35 of SEQ ID NO:2, andpreferably amino acids 2345 of SEQ ID NO: 2. In still anotherembodiment, a fragment includes amino acids 138-155 of SEQ ID NO:2 andcomprises at least 19 amino acids in length. In still anotherembodiment, a fragment includes amino acids 179-336, or a smallerfragment of at least 8 amino acids contained therein. Still otherfragments may be selected from the sequence. In yet another embodiment,the invention provides analogs or homologs of SEQ ID NO:2. In stillanother embodiment, the invention provides a fusion protein comprisingthe amino acid sequence of SEQ ID NO: 2, a fragment, analog or homologthereof, and a fusion partner. In still a further embodiment, theinvention provides a deletion protein comprising the amino acid sequenceof SEQ ID NO:2 with one to twenty amino acids deleted therefrom.

In another aspect, the present invention provides a Bin2 nucleic acidsequence. Desirably, the Bin2 nucleic acid sequence encodes a protein orfragment of the invention (such as those mentioned above) and containsSEQ ID NO:1 or a fragment thereof. In one embodiment, the Bin2 nucleicacid sequence hybridizes to the sequence of SEQ ID NO:1 under stringentconditions. In another embodiment, the invention provides a nucleic acidsequence complementary to the nucleic acid sequence of SEQ ID NO:1. Instill another embodiment, the invention provides a nucleic acid sequenceencoding a fusion protein of the invention. In a further embodiment, theinvention provides an allelic variant of any of the Bin2 nucleic acidsequences of the invention. In still another embodiment, the nucleicacid sequence is an antisense sequence to the sequences described above.

In a further aspect, the invention provides a vector comprising a Bin2nucleic acid sequence of the invention under the control of regulatorysequences which direct expression of the Bin2 protein.

In still another aspect, the invention provides a host cell transformedwith the vector of the invention.

In yet a further aspect, the invention provides a diagnostic reagentcomprising a Bin2 nucleic acid sequence of the invention and adetectable label which is associated with said sequence. Methods ofdiagnosing conditions associated with inappropriate functional levels,the loss of expression of Bin2 or altered expression of Bin2, e.g.,cancers, which use this reagent are also provided.

In still a further aspect, the invention provides a diagnostic reagentcomprising a Bin2 protein or peptide of the invention and a detectablelabel which is associated with that protein. Also provides are methodsof using this reagent and/or the Bin2 protein for diagnosing cancersassociated with inappropriate expression (e.g., overexpression orunderexpression or altered expression) of Bin1, to which Bin2 binds.This method involves the steps of contacting a sample from a human oranimal to be diagnosed with the Bin2 protein of the invention, or thediagnostic reagent containing this protein, whereby in the presence ofBin1 in the sample, a complex is formed between Bin1 and the Bin2protein or reagent, and analyzing for the presence of said complex.

In yet another aspect, the invention provides an isolated anti-Bin2antibody which is specific for the Bin2 protein of the invention.

In still another aspect, the invention provides a diagnostic reagentcomprising the anti-Bin2 antibody of the invention and a detectablelabel. Further provided by the invention is a method of diagnosingcancer or hyperplastic disease characterized by inappropriate levels oraltered expression of functional Bia2 in a human or an animal using theanti-Bin2 antibody or diagnostic reagent of the invention. This methodinvolves contacting an anti-Bin2 antibody or a diagnostic reagentcontaining same with a sample from a human or animal to be diagnosed,whereby in the presence of Bin2, a detectable complex is formed with theBin2 protein or diagnostic reagent, analyzing for the presence orabsence of said complex; and comparing the level of complex to astandard, wherein the absence of said detectable label indicates theabsence of functional Bin2.

In still another aspect, the invention provides a kit for diagnosing acondition associated with Bin2 comprising a diagnostic reagent of theinvention.

In a further aspect, the invention provides an anti-idiotype antibodyspecific for the anti-Bin2 antibody of the invention.

In yet another aspect, the invention provides a composition comprisingan effective amount of a Bin2 protein or anti-idiotype of the inventionand a pharmaceutically acceptable carrier.

In still another aspect, the invention provides a method of detectinginappropriate expression of Box dependent myc-interacting protein-2(Bin2) in a patient comprising providing a sample from a patientsuspected of having said inappropriate (over- or under-expression) oraltered expression; incubating said sample in the presence of ananti-Bin2 antibody or a diagnostic reagent containing same; andcomparing levels of expression to a normal Bin2 control.

In a further aspect, the invention provides a method of detectinginappropriate expression of Box-dependent myc-interacting peptide-2Bin2) in a patient comprising providing a sample from a patent suspectedof having said inappropriate expression and performing nucleic acidamplification using a Bin2 nucleic acid sequence of the invention.

In still a further aspect, the invention provides a method ofidentifying compounds which specifically bind to Bin2 or whichspecifically inhibit or block the binding of Bin2 to its ligand. In oneembodiment, the method involves comprising the steps of contacting saidBin2 or a fragment thereof with a test compound to permit binding of thetest compound to Bin2; and determining the amount of test compound whichis bound to Bin2. In another embodiment, the invention provides a methodof contacting an amount of immobilized first Bin peptide or a fragmentthereof with a test compound and an amount of labeled second Bin peptideor fragment, wherein said first Bin peptide is either a Bin1 or a Bin2peptide, and the second Bin peptide is the Bin peptide that binds saidfirst Bin peptide. Unbound material is separated from immobilizedmaterial and the amount of label on said immobilized material ismeasured. A decrease in the amount of label immobilized in the presenceof test compound compared to the amount of label immobilized in thepresence of a control peptide or protein, indicates that said testcompound inhibits the binding of Bin1 to Bin 2, or vice versa. Theinvention further provides a compound identified by this method.

Other aspects and advantages of the present invention are describedfurther in the following detailed description of the preferredembodiments thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C illustrate the continuous sequence of full-length Bin2 cDNA[SEQ ID NO: 1] and the continuous amino acid sequence encoded thereby[SEQ ID NO: 2]. The initiating methionine codon is underlined and thestop codon TCT is indicated by an asterisk 5′ and 3′ noncoding sequencesare shown.

FIG. 2 provides an alignment of the BAR domains of Bin2 [SEQ ID NO: 2]and Bin1 [SEQ ID NO: 3].

FIG. 3 is an illustration comparing Bin2 structure with other BAR familymembers.

FIG. 4A provides the amino acid sequence alignment of Bin1 [SEQ ID NO:

3], Bin2 [SEQ ID NO: 2], and Daxn [SEQ ID NO: 4].

FIG. 4B is an illustration of the domains in Bin1, Bin2, and Daxx andthe relative region of similarity between Bin2 and Daxx (dark shaded).

FIG. 5A is a bar graph illustrating the results of a colony formationassay. HepG2 and A549 cells were transfected with expression vectors andstable transformants were selected by culturing cells in G418. Colonieswere scored by methanol fixation and crystal violet staining 2-3 weekslater. The data represent the mean and standard error from three trials.

FIG. 5B is a bar graph that illustrates that Bin2 does not affect thetumor suppressor activity of Bin1. The colony formation assay wasperformed as above using 10 μg vector, 5 μg vector +5 μg Bin1 plasmid,or 5 μg Bin1+5 μg Bin2 plasmids.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel, isolated mammalian Bin nucleicacid sequences, fragments thereof and proteins and peptides encodedthereby. The invention further provides methods of using thesesequences, proteins, and compositions containing them for diagnosis andtreatment of disorders associated with deregulation, deficiency oramplification of the c-myc oncogenes. The present invention furtherprovides methods of using these sequences to generate antibodies and newcompositions useful for such diagnosis and treatments.

The present invention provides Bin2 (Bridging INtegator-2), which is anovel member of the BAR family. Bin2 is expressed predominantly inhematopoietic cells and can form a stable complex with Bin1. Bin2 isupregulated during differentiation of granulocytes, thereby functioningin that cell lineage. Bin2 lacks sequences found in Bin1 that mediatec-Myc interaction. Bin2 also lacks sequences found in amphiphysin thatmediate interaction with endocytotic machinery in the brain. Inaddition, Bin2 lacks a C-terminal region SH3 domain, instead including aC-terminal extension that is unrelated to other members of the BARfamily. Database comparisons with Bin2 revealed a previouslyunrecognized region of similarity between BAR family proteins and Daxx,a nucleocytoplasmic adaptor implicated in programmed cell death, JNKsignaling, and chromosomal regulation [Chang et al., Science 281:1860-1863 (1998); Pluta et al., J. Cell Sci. 111: 2029-2041 (1998); Yanget al., Cell 89: 1067-1076 (1997)], within the central part of theseproteins. The human Bin2 gene was mapped to chromosome 4q22.1, within aregion that is frequently deleted in breast and liver cancers. However,unlike Bin1, which is ubiquitous and growth inhibitory, Bin2 wasexpressed predominantly in hemapoietic cells and was found to lackdetectable antiproliferative activity. Thus, Bin2 is a novel BAR proteinwhich has nonredundant functions relative to other members of the BARfamily.

Compositions containing Bin2 proteins and nucleic acid sequences areuseful for a variety of purposes. These aspects of the invention arediscussed in more detail below.

I. Nucleic Acid Sequences

The present invention provides mammalian nucleic acid sequences encodinga Bin2 protein or peptide. The nucleic acid sequences of this inventionmay be isolated from cellular materials with which they are naturallyassociated or produced using techniques known in the art. In oneembodiment, the present invention provides Bin2 nucleic acid sequenceSEQ ID NO:1 and fragments of at least eight contiguous amino acidsthereof. However, the present invention is not limited to these nucleicacid sequences.

Given the sequences of the Bin2 DNA [SEQ ID NO:1], one of skill in theart can readily obtain the corresponding anti-sense strands to thesesequences. Further, using known techniques, one of skill in the art canreadily obtain further sequences, including cDNA sequences or thecorresponding RNA sequences, as desired.

Allelic variants of these sequences within a species (i.e., sequencescontaining some individual nucleotide differences from a more commonlyoccurring sequence within a species, but which nevertheless encode thesame protein or a protein with the same function) may also be readilyobtained given the knowledge of the nucleic acid sequence provided bythis invention.

The present invention further encompasses nucleic acid sequences capableof hybridizing under stringent conditions [see, J. Sambrook et al,Molecular Cloning A Laboratory Manual, 2d ed., Cold Spring HarborLaboratory (1989)] to the sequences of the invention, SEQ ID NO:1, theiranti-sense strands, or biologically active fragments thereof. An exampleof a highly stringent hybridization condition is hybridization at 2×SSCat 65° C., followed by a washing in 0.1×SSC at 65° C. for an hour.Alternatively, an exemplary highly stringent hybridization condition isin 50% formamide, 4×SSC at 42° C. Moderately high stringency conditionsmay also prove useful, e.g. hybridization in 4×SSC at 55° C., followedby washing in 0.1×SSC at 37° C. for an hour. An alternative exemplarymoderately high stringency hybridization condition is in 50% formamide,4×SSC at 30° C.

According to the invention, the nucleic acid sequences may be modified.Utilizing the sequence data provided herein, it is within the skill ofthe art to obtain or prepare synthetically or recombinantly otherpolynucleotide sequences, or modified polynucleotide sequences, encodingthe full-length proteins or useful fragments of the invention. Suchmodifications at the nucleic acid level include, for example,modifications to the nucleotide sequences which are silent or whichchange the amino acids, e.g. to improve expression or secretion. Alsoincluded are allelic variations, caused by the natural degeneracy of thegenetic code.

Also encompassed by the present invention are nucleotide sequencesencoding mutants of the Bin2 peptides and proteins provided herein. Suchmutants include amino terminal, carboxy terminal or internal deletions,which substantially retain the biological activity (e.g., theantigenicity and/or the ability to bind Bin1) of the full-length Bin2 orother proteins or fragments. Such a truncated, or deletion mutant may beexpressed for the purpose of affecting the activity of the full-lengthor wild-type gene or gene fragments.

Thus, the invention provides nucleic acid sequence fragments that encodea desirable fragment of Bin2, e.g., a Bin1 binding region. Generally,these oligonucleotide fragments are at least 15 nucleotides in length.However, oligonucleotide fragments of varying sizes may be selected asdesired. Such fragments may be used for such purposes as performingpolymerase chain reaction (PCR), e.g., on a biopsied tissue sample. Forexample, suitable nucleic acid fragments include those encoding thecomplete BAR motif or domain (amino acid 1 to 221 or amino acid 1 to 249of SEQ ID NO:2) and the putative BAR effector region (amino acid 138-155of SEQ ID NO:2). In one embodiment a fragment of this invention encodesat least 8 contiguous amino acids in length, e.g., a fragment selectedfrom amino acids 1 to 13 of SEQ ID NO:2. In another embodiment, anucleic acid sequence of this invention encodes at least 14 amino acidsin length. Exemplary fragments include those encoding amino acids 23 to35 or a peptide within amino acids 23 to 45 of SEQ ID NO:2. In stillanother embodiment, a nucleic acid sequence encodes a BIN2 fragmentwithin amino acids 138-155 of SEQ ID NO:2 and comprises at least 19amino acids in length. Still another nucleic acid sequence of theinvention encodes a peptide of at least 8 amino acids in length withinamino acids 179 to 336 of SEQ ID NO: 2. Still another nucleic acidsequence of the invention encodes a peptide of at least 8 amino acids inlength within amino acids 250 to 564 of SEQ ID NO: 2, which is theregion unrelated to the other BAR family proteins. In yet anotherembodiment, the nucleotide sequences of the invention encode analogs orhomologs of SEQ ID NO:2. Instill another embodiment, the nucleotidesequences of this invention encode fusion proteins comprising the aminoacid sequence of SEQ ID NO: 2, a fragment, analog or homolog thereof,and a fusion partner. In still a further embodiment, a nucleotidesequence of this invention encodes a deletion protein comprising theamino acid sequence of SEQ ID NO:2 with one to twenty amino acidsdeleted therefrom. Other useful fragments may be readily identified byone of skill in the art by resort to conventional techniques.

The nucleotide sequences of the invention may be isolated byconventional uses of polymerase chain reaction or cloning techniquessuch as those described in obtaining the murine and human sequences,described below. Alternatively, these sequences may be constructed usingconventional genetic engineering or chemical synthesis techniques.

These nucleic acid sequences are useful for a variety of diagnostic,prophylactic and therapeutic uses. Advantageously, the nucleic acidsequences are useful in the development of diagnostic probes andantisense probes for use in the detection and diagnosis of cancers andother conditions associated with inappropriate levels of functional Bin2(and/or its binding partner, Bin1) or altered expression of Bin2 byutilizing a variety of known nucleic acid assays, e.g., Northern andSouthern blots, polymerase chain reaction (PCR), and other assaytechniques known to one of skill in the art. When used in diagnosticapplications, the nucleic acid sequences of the invention may optionallybe associated with a detectable label, such as are described in detailbelow. The nucleic acid sequences of this invention are also useful inthe production of the peptides and proteins of the invention in vitro,in vivo, and ex vivo.

II. Protein Sequences

The present invention also provides mammalian Bin2 polypeptides orproteins. For convenience throughout this specification, reference willbe made to “Bin2 peptides and proteins”, but it will be understood thatthis tern encompasses the fragments, analogs, modified peptides andproteins, fusion proteins, and other amino acid constructs of theinvention, except where otherwise specified.

These Bin2 peptides and proteins may be isolated in a form substantiallyfree from other proteinaceous and non-proteinaceous cellular materials(e.g., hepatocytes) or from cell extracts. For example, these peptidesand proteins may be isolated from the cellular materials and optionally,further purified using any of a variety of conventional methodsincluding: liquid chromatography such as normal or reversed phase, usingHPLC, FPLC and the like; affinity chromatography (such as with inorganicligands or monoclonal antibodies); size exclusion chromatography;

immobilized metal chelate chromatopraphy; gel electrophoresis, and thelike. One of skill in the art may select the most appropriate isolationand purification techniques without departing from the scope of thisinvention.

Alternatively, the Bin2 peptides and proteins of the invention,described below, may be produced recombinantly following conventionalgenetic engineering techniques [see e.g., Sambrook et al, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratories, ColdSpring Harbor, N.Y. and the detailed description of making the proteinsbelow]. In still another alternative, the peptides and proteins of theinvention may be produced using conventional chemical synthesistechniques, such as those described in G. Barony and R. B. Merrifield,The Peptides: Analysis, Synthesis & Biology, Academic Press, pp. 3-285(1980), among others. The term “artificial” is used herein to denote thepreparation of the construct (e.g., a peptide, protein, nucleic acid, orantibody of the invention) by chemical synthesis, recombinanttechnology, or other similar means.

The present invention further provides analogs, fragments, and mutantpeptides, as well as proteins containing Bin2, or such analogs,fragments or mutants, as described below.

A. Analogs and Modified Peptide and Protein Antigens

Analogs or modified versions of the Bin2 proteins and peptides areprovided. Typically, analogs differ from the specifically identifiedproteins by only one to four codon changes. Examples includepolypeptides with minor amino acid variations from the illustrated aminoacid sequence of Bin2 having conservative amino acid replacements.Conservative replacements are those that take place within a family ofamino acids that are related in their side chains and chemicalproperties. Also provided are homologs of the proteins of the inventionwhich are characterized by having at least 90% identity, and morepreferably 95-99% identity with Bin2 sequences. Based on the sequenceinformation provided herein, one of skill in the art can readily obtainfull-length homologs and analogs.

As known in the art, “homology” or “identity” means the degree ofsequence relatedness between two peptide or two nucleotide sequences asdetermined by the identity of the match between two lengths of suchsequences. Both identity and homology can be readily calculated bymethods extant in the prior art [See, e.g., COMPUTATIONAL MOLECULARBIOLOGY, Lesk, A. M., ed., Oxford University Press, New York, (1988);BIOCOMPUTING: INFORMATICS AND GENOME PROJECTS, Smith, D. W., ed.,Academic Press, New York, (1993); COMPUTER ANALYSIS OF SEQUENCE DATA,PART I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, NewJersey, (1994); SEQUENCE ANALYSIS IN MOLECULAR BIOLOGY, von Heinje, G.,Academic Press, (1987); and SEQUENCE ANALYSIS PRIMER, Gribskov, M. andDevereux, J., eds., M Stockton Press, New York, (1991)]. While thereexist a number of methods to measure identity and homology between twonucleotide sequences, the terms “identity”, “similarity” and “homolog”are well known to skilled artisans [H. Carillo and D. Lipton, SIAM J.Applied Math., 48:1073 (1988)]. Methods commonly employed to determineidentity or homology between two sequences include, but are not limitedto, those disclosed in GUIDE TO HUGE COMPUTERS, Martin J. Bishop, ed.,Academic Press, San Diego, 1994. Preferred methods to determine identityor homology are designed to give the largest match between the twosequences tested. Methods to determine identity and similarity arecodified in computer programs. Preferred computer program methods todetermine identity and homology between two sequences include, but arenot limited to, the algorithm BESTFIT from the GCG program package [J.Devereux et al., Nucl. Acids Res., 12(1):387 (1984)], the relatedMACVECTOR program (Oxford), and the FASTA (Pearson) programs, which maybe used at default settings or modified settings such as determined tobe suitable by one of skill in the art.

A Bin2 peptide or protein of the present invention may also be modifiedto increase its ability to bind and thus, complex with, Bin1. Forexample, the Bin2 peptide or protein may be coupled to chemicalcompounds or non-proteinaceous carriers. In certain embodiments, thecoupling is designed not to interfere with the desired biologicalactivity of either the Bin2 peptide or protein or the carrier. For areview of some general considerations in coupling strategies, seeAntibodies, A Laboratory Manual, Cold Spring Harbor Laboratory, ed. E.Harlow and D. Lane (1988). For example, the carrier may be selectedwhich facilitates cell penetration, e.g. a lipid or a carbohydrate. Asanother example, the carrier may be selected to deliver a toxin to Bin1,to which the Bin2 peptide binds. Such toxins are known to those of skillin the art and may include, e.g., chemical compounds including, withoutlimitation, dinitrophenol groups and arsonilic acid. Yet other carriersmay be selected simply to facilitate production or delivery of the Bin2peptide or protein. For example, useful carriers known in the art,include, without limitation, keyhole limpet hemocyanin (KLH); bovineserum albumin (BSA), ovalbumin, agarose beads; activated carbon; orbentonite.

The Bin2 peptides and proteins of the invention may also be modified byother techniques, such as by denaturation with heat and/or SDS.Alternatively, the peptides and proteins of the invention may bemodified to provide an additional N- or C-terminal amino acid sequencesuitable for biotinylation, e.g., cysteine or lysine.

B. Fragments Deletion Mutants

Further encompassed by this invention are additional fragments of theBin2 peptide or of the other proteins identified herein. Such fragmentsare desirably characterized by having a biological activity similar tothat displayed by the complete protein, including, e.g., the ability tobind and complex with Bin1. These fragments may be designed or obtainedin any desired length, including as small as about 5 to about 8 aminoacids in length, about 14 or 15 amino acids in length, about 19 to 20amino acids in length, or longer. Such a fragment represents less thanthe full-length Bin2 protein and may represent as little as a singleepitope of the protein.

For example, one particularly desirable fragment of the invention is theBAR domain (amino acid 1 to 221 or amino acid 1 to 249 of SEQ ID NO:2),which contains dimerization signals. Optionally, one of skill in the artmay utilize fragments with the BAR domain, which fragments are unique toBIN2, yet exhibit the desired biological function. Suitably, fragmentsof the BAR domain are composed of at least 14 amino acids in length withrespect to any fragment which encompasses all of amino acids 1 to 13 orwhich encompasses all of amino acids 23 to 35 or amino acids 23 to 45 ofSEQ ID NO:2. Another desirable fragment encompasses the putative BAReffector region (amino acid 138 to 155 of SEQ ID NO:2), which isimplicated in Bin1 in tumor suppressor and programmed cell deathsignaling. Still other desirable fragments include fragments of about 8or more amino acids from amino acids 250 to 564 of SEQ ID NO: 2, the Cterminal region unrelated to other BAR family proteins. Suitably, anyfragments of Bin2 containing these latter regions are at least 19 aminoacids in length.

In yet another example, a Bin2 fragment may be a T cell epitope. Such aT cell epitope may be readily identified using available computermodeling programs.

Optionally, the peptides of the invention may be modified to createdeletion mutants, for example, by truncation at the amino or carboxytermini, or by elimination of one or more amino acids. Still othermodified fragments of Bin2 may be prepared by any number of nowconventional techniques to improve production thereof, to enhanceprotein stability or other characteristics, e.g. binding activity orbloavailability, or to confer some other desired property upon theprotein. Other useful fragments of these polypeptides may be readilyprepared by one of skill in the art using known techniques, such asdeletion mutagenesis and expression.

C. Fusion or Multimeric Proteins and Compositions

The Bin2 peptides and proteins of the present invention, or fragments ofthem, may also be constructed, using conventional genetic engineeringtechniques as part of a larger and/or multimeric protein or proteincomposition.

For example, such a fusion protein may be desirable in order to improveyield on expression and/or purification. Suitable fusion partners forsuch a purpose are well known to those of skill in the art and include,e.g., glutathione-S-transferase and maltose binding protein.Alternatively, a fusion protein of the invention may be composed of aBin2 fragment, such as a fragment corresponding to a T cell epitope orto the Bin1 binding region, which is fused to an active agent.

The active agent may be composed of other Bin2 peptides and proteins ofthis invention, or may be other proteinaceous materials or nucleic acidmolecule. For example, it may be desirable to fuse a Bin2 peptide orprotein of the invention with a proteinaceous molecule which facilitatesits cell penetration, e.g., Drosophila antennapedia, HIV Rev peptides,which are known to those of skill in the art. Alternatively, it may bedesirable to fuse a Bin2 peptide or protein of the invention to amolecule which is to be targeted to a particular cell type and/ortoBin1. Particularly suitable are toxins and anti-cancer agents, any ofwhich are known to those of skill in the an. However, suitable examplesinclude, without limitation, tetanus toxoid, cholera toxoid, PPD(purified protein derivative of tuberculin), and molecular toxins suchas imidazole protein cross-linkers or other conjugates (which would killa bound Bin1 molecule). These proteins are effective in the prevention,treatment and diagnosis of cancers associated with inappropriate levelsof functional Bin2 and/or inappropriate Bin1 function.

The fusion proteins of the invention are constructed for use in themethods and compositions of this invention. These fusion proteins may beproduced recombinantly, or may be synthesized chemically. A proteincomposition which may be a preferred alternative to the fusion proteinsdescribed above is a cocktail (i.e., a simple mixture) containing a Bin2peptide or protein, or different mixtures of the Bin2 peptides andproteins of this invention. In still another aspect, the peptide andproteins of the invention may be provided with a detectable label, suchas are described in detail below.

D. Salts

A peptide or protein antigen of the present invention may also be usedin the form of a pharmaceutically acceptable salt. Suitable acids andbases which are capable of forming salts with the polypeptides of thepresent invention are well known to those of skill in the art, andinclude inorganic and organic acids and bases.

III. Methods of Making Proteins and Nucleic Acid Sequences of theInvention Expression

The proteins and nucleic acid molecules of the invention may be isolatedfrom natural sources as described above, or may be produced usingchemical synthesis techniques, such as are well known to those of skillin the art. In still another alternative, the peptides and proteins ofthe invention may be produced using conventional chemical synthesistechniques, such as those described in G. Barony and R. B. Merrifield,THE PEPTIDES: ANALYSIS, SYNTHESIS & BIOLOGY, Academic Press, pp. 3-285(1980), among others. Particularly desirable, is the use of recombinanttechnology to produce the proteins and nucleic acid sequences of theinvention.

A. Expression In Vitro

To produce recombinant Bin2 proteins of this invention, a DNA sequenceof the invention is inserted into a suitable expression system.Desirably, a recombinant molecule or vector is constructed in which thepolynucleotide sequence encoding Bin2 is operably linked to aheterologous expression control sequence permitting expression of theBin2 protein. Numerous types of appropriate expression vectors are knownin the art for protein expression, by standard molecular biologytechniques. Such vectors may be selected from among conventional vectortypes including insects, e.g., plasmids, yeast, fungal, bacterial,insect (e.g., baculovirus expression) or viral expression systems. Otherappropriate expression vectors, of which numerous types are known in theart, can also be used for this purpose. Methods for obtaining suchexpression vectors are well-known. See, Sambrook et al, MOLECULARCLONING. A LABORATORY MANUAL, 2d edition, Cold Spring Harbor Laboratory,New York (1989); Miller et al, Genetic Engineering, 8:277-298 (PlenumPress 1986) and references cited therein.

Suitable host cells or cell lines for transfection by this methodinclude mammalian cells, such as human 293 cells, Chinese hamster ovarycells (CHO), the monkey COS-1 cell line or murine 3T3 cells derived fromSwiss, Balb-c or NIH mice may be used. Another suitable mammalian cellline is the CV-1 cell line. Still other suitable mammalian host cells,as well as methods for transfection, culture, amplification, screening,production, and purification are known in the an. [See, e.g., Gethingand Sambrook, Nature, 293:620-625 (1981), or alternatively, Kaufman etal, Mol. Cell. Biol., 5(7):1750-1759 (1985) or Howley of al, U.S. Pat.No. 4,419,446].

Similarly, bacterial cells are useful as host cells for the presentinvention. For example, the various strains of E. coli (e.g., FIB101,MC1061, and strains used in the following examples) are well-known ashost cells in the field of biotechnology. Various strains of B.subtilis, Pseudomonas, other bacilli and the like may also be employedin this method. Many strains of yeast cells known to those skilled inthe art are also available as host cells for expression of thepolypeptides of the present invention. Other fungal cells may also beemployed as expression systems. Alternatively, insect cells such asSpodoptera frugipedera (Sf9) cells may be used, e.g., in the baculovirusexpression system. Alternatively, insect cells such as Spodopterafrugipedera (Sf9) cells may be used, e.g., in the baculovirus expressionsystem.

Thus, the present invention provides a method for producing arecombinant Bin2 protein which involves transfecting a host cell with atleast one expression vector containing a recombinant polynucleotideencoding a Bin2 protein under the control of a transcriptionalregulatory sequence, e.g., by conventional means such aselectroporation. The transfected host cell is then cultured underconditions that allow expression of the Bin2 protein. The expressedprotein is then recovered, isolated, and optionally purified from theculture medium (or from the cell, if expressed intracellularly) byappropriate means known to one of skill in the art.

For example, the proteins may be isolated in soluble form following celllysis, or may be extracted using known techniques, eg., in guanidinechloride. If desired, the Bin2 proteins of the invention may be producedas a fusion protein. For example, it may be desirable to produce Bin2fusion proteins, to enhance expression of the protein in a selected hostcell, to improve purification, or for use in monitoring the presence ofBin2 in cells, e.g., hepatocytes, or cell extracts. Suitable fusionpartners for the Bin2 proteins of the invention are well known to thoseof skill in the art and include, among others, β-alactosidase,glutatliione-S-transferase, and poly-histidine.

B. Expression In Vivo

Alternatively, where it is desired that the Bin2 protein be expressed invitao, e.g., for therapeutic purposes, an appropriate vector fordelivery of Bin2, or fragment thereof, may be readily selected by one ofskill in the art. Exemplary vectors are readily available from a varietyof academic and commercial sources, and include, e.g., DNA vectors(including “naked” DNA and plasmid systems), adeno-associated virus,adenovirus vectors, or other viral vectors, e.g., various poxviruses,vaccinia, etc. Methods for insertion of a desired gene, e.g. Bin2, andobtaining in vivo expression of the encoded protein, are well known tothose of skill in the art.

IV. Antibodies of the Invention

The Bin2 proteins of this invention are also useful as antigens for thedevelopment of anti-Bin2 antisera and antibodies to Bin2 or to a desiredfragment of a Bin2 protein. Specific antisera may be generated usingknown techniques. See, Sambrook, cited above, Chapter 18, generally,incorporated by reference. Similarly, antibodies of the invention, bothpolyclonal and monoclonal, may be produced by conventional methods,including the Kohler and Milstein hybridoma technique and the many knownmodifications thereof. Similarly desirable antibodies are generated byapplying known recombinant techniques to the monoclonal or polyclonalantibodies developed to these antigens [see, e.g., PCT PatentApplication No. PCT/GB85/00392 British Patent Application PublicationNo. GB2188638A; Amit et al., Science, 233:747-753 (1986); Queen et at.,Proc. Nat'l. Acad. Sci. USA, 86:10029-10033 (1989); PCT PatentApplication No. PCT/WO9007861; and Riechmann et al. Nature, 332:323-327(1988); Huse el a/, Science, 246:1275-1281 (1988)], or any othertechniques known to the art.

Given the disclosure contained herein, one of skill in the art maygenerate chimeric, humanized or fully human antibodies directed againsta Bin2 peptide or protein of the invention by resort to known techniquesby manipulating the complementarity determining regions of animals orhuman antibodies to the Bin2 protein of the invention. See, e.g., E.Mark and Padlin, “Humanization of Monoclonal Antibodies”, Chapter 4, THEHANDBOOK OF EXPERIMENTAL PHARMACOLOGY, Vol. 113, The Pharmacology ofMonoclonal Antibodies, Springer-Verlag (June, 1994). Alternatively, theantigens may be assembled as multi-antigenic complexes [see, e.g.,European Patent Application 0339695, published Nov. 2, 1989] or assimple mixtures of antigenic proteins/peptides and employed to elicithigh titer antibodies capable of binding the selected antigen(s) as itappears in the biological fluids of an infected animal or human.

Further provided by the present invention are anti-idiotype antibodies(Ab2) and anti-anti-idiotype antibodies (Ab3). Ab2 are specific for thetarget to which anti-Bin2 antibodies of the invention bind and Ab3 aresimilar to Bin2 antibodies (Ab1) in their binding specificities andbiological activities [see, e.g., M. Wettendorffet al., “Modulation ofanti-tumor immunity by anti-idiotypic antibodies.” In IDIOTYPIC NETWORKAND DISEASES, ed. by J. Cerny and J. Hiernaux J, Am. Soc. Microbiol.,Washington D.C.: pp. 203-229, (1990)]. These anti-idiotype andanti-anti-idiotype antibodies may be produced using techniques wellknown to those of skill in the art. Such anti-idiotype antibodies (Ab2)can bear the internal image of Bin1 and bind to it in much the samemanner as Bin2 and are thus useful for the same purposes as Bin2.

In general, polyclonal antisera, monoclonal antibodies and otherantibodies which bind to Bin2 as the antigen (Ab1) are useful toidentify epitopes of Bin2, to separate Bin2 from contaminants in livingtissue (e.g., in chromatographic columns and the like), and in generalas research tools and as starting material essential for the developmentof other types of antibodies described above. Anti-idiotype antibodies(Ab2) are useful for binding Bin2 and thus may be used in the treatmentof cancers in which Bin2 is part of a biochemical cascade of eventsleading to carcinoma. The Ab3 antibodies may be useful for the samereason the Ab1 are useful. Other uses as research tools and ascomponents for separation of Bin2 from other contaminants of livingtissue, for example, are also contemplated for these antibodies.

For use in diagnostic assays, the antibodies are associated withconventional labels which are capable, alone or in concert with othercompositions or compounds, of providing a detectable signal. Where morethan one antibody is employed in a diagnostic method, the labels aredesirably interactive to produce a detectable signal. Most desirably,the label is detectable visually, e.g. colorimetrically. A variety ofenzyme systems have been described in the art which will operate toreveal a colorimetric signal in an assay. As one example, peroxidase,which reacts with peroxide and a hydrogen donor such as tetramethylbenzidine (TMB), produces an oxidized IMB that is seen as a blue color.Other examples include horseradish peroxidase (HRP) or alkalinephosphatase (AP), and hexokinase in conjunction with glucose-6-phosphatedehydrogenase which reacts with ATP, glucose, and NAD+ to yield, amongother products, NADH that is detected as increased absorbance at 340 nmwavelength. Still another example is glucose oxidase (which uses glucoseas a substrate) which releases peroxide as a product. Other labelsystems that may be utilized in the methods of this invention aredetectable by other means, e.g., colored latex microparticles [BangsLaboratories, Indiana] in which a dye is embedded may be used in placeof enzymes to form conjugates with the antibodies and provide a visualsignal indicative of the presence of the resulting complex in applicableassays. Still other labels include fluorescent compounds, radioactivecompounds or elements. Detectable labels for attachment to antibodiesuseful in diagnostic assays of this invention may be easily selectedfrom among numerous compositions known and readily available to oneskilled in the art of diagnostic assays. The methods and antibodies ofthis invention are not limited by the particular detectable label orlabel system employed. Suitably, these detectable systems may also beutilized in connection with diagnostic reagents composed of thepeptides, proteins, and nucleic acid sequences of the invention.

V. Diagnostic Reagents and Methods

Advantageously, the present invention provides reagents and methodsuseful in detecting and diagnosing a deficiency in normal Bin2 levelsand/or abnormal levels of non-functional Bin2, and particularlydeficiencies or excess production thereof, in a patient. Further, thepresent invention provides reagents which bind Bin1, and therefore asdefined herein, a deficiency of Bin2 is an inadequate level offunctional Bin2 to compensate for the levels of Bin1 in a patient. Adeficiency of Bin1 is an inadequate level of functional Bin1 tocompensate for the levels of c-Myc in a patient. Conditions associatedwith deficiencies of Bin2 include hepatocarcinoma; conditions associatedwith Bin1 include a variety of cancers, e.g., epithelial cell cancer,breast cancer, melanoma, prostate cancer, liver cancer and colon cancer,and hyperplastic disease states, e.g., benign prostate hyperplasia.Conditions associated with altered expression or loss of expression ofnormal Bin2 include myeloid and lymphoid leuklemias.

For convenience, reference will be made to Bin2 proteins throughout thisand the following section. However, it will be understood that Bin2nucleic acids (including anti-sense sequences and oligonucleotidefragments, among others), peptides, analogs and diagnostic compositionscontaining these molecules may be useful in these methods.

In one embodiment, this method involves detecting the presence of Bin1(or other ligand for Bin2) which is produced by the affected human oranimal patient's system and which are capable of binding to the Bin2peptides and proteins (or Ab2) of this invention or combinationsthereof. This method comprises the steps of incubating a Bin2 peptide orprotein of this invention with a sample of biological fluids from thepatient. Bin1 present in the samples will form a complex with the Bindpeptide or protein. Subsequently the reaction mixture is analyzed todetermine the presence or absence of these complexes. The step ofanalyzing the reaction mixture comprises contacting the reaction mixturewith a labeled specific binding partner for the Bin2 ligand.

In one embodiment of the method, the Bin2 peptide or protein, or amixture of the peptides and proteins of the invention is electro- ordot-blotted onto nitrocellulose paper. Subsequently, the biologicalfluid (e.g. serum or plasma) is incubated with the blotted peptide orprotein, and ligand (e.g., Bin1) in the biological fluid is allowed tobind to the Bin2 peptide or protein. The bound ligand is then detectedby standard immunoenzymatic methods.

In another embodiment of the method, latex beads are conjugated to theBin2 peptide or protein of this invention. Subsequently, the biologicalfluid is incubated with the bead/protein conjugate, thereby forming areaction mixture. The reaction mixture is then analyzed to determine thepresence of Bin1 or other Bin2 ligand.

In another embodiment, the diagnostic method of the invention involvesdetecting the presence of the naturally occurring Bin2 peptide orprotein itself in its association with hepatocytes in the biologicalfluids of an animal or human infected by the pathogen. This methodincludes the steps of incubating a ligand specific for Bin2 (e.g., Bin1or an antibody of this invention, e.g. produced by administering to asuitable human and/or animal an antigen of this invention), preferablyconventionally labelled for detection, with a sample of biologicalfluids from a human or an animal to be diagnosed. In the presence ofBin2, a complex is formed (specific binding occurs). Subsequently,excess labeled antibody (or other ligand) is optionally removed, and thereaction mixture is analyzed to determine the presence or absence of theantigen-antibody complex and the amount of label associated therewith.

Assays employing a peptide or protein of the invention can beheterogenous (i.e., requiring a separation step) or homogenous. If theassay is heterogenous, a variety of separation means can be employed,including ccntrifugation, filtration, chromatography, or magnetism.

One preferred assay for the screening of blood products or otherphysiological or biological fluids is an enzyme linked immunosorbantassay, i.e., an ELISA. Typically in an ELISA, the isolated Bin2 peptideor protein of the invention is adsorbed to the surface of a microtiterwell directly or through a capture matrix (i.e., antibody). Residualprotein-binding sites on the surface are then blocked with anappropriate agent, such as bovine serum albumin (BSA), heat-inactivatednormal goat serum (NGS), or BLOTTO (a buffered solution of nonfat drymilk which also contains a preservative, salts, and an antifoamingagent). The well is then incubated with a biological sample suspected ofcontaining Bin1 or another ligand specific for Bin2. The sample can beapplied neat, or more often, it can be diluted, usually in a bufferedsolution which contains a small amount (0.1-5.0% by weight) of protein,such as BSA, NGS, or BLOTTO. After incubating for a sufficient length oftime to allow specific binding to occur, the well is washed to removeunbound protein and then incubated with labeled anti-humanimmunoglobulin (a Hulg) or labeled antibodies to other species, e.g.,dogs. The label can be chosen from a variety of enzymes, includinghorseradish peroxidase (HRP), β-galactosidase, alkaline phosphatase, andglucose oxidase, as described above. Sufficient time is allowed forspecific binding to occur again, then the well is washed again to removeunbound conjugate, and the substrate for the enzyme is added. Color isallowed to develop and the optical density of the contents of the wellis determined visually or instrumentally.

Further, MAbs or other antibodies of this invention which are capable ofbinding to Bin2 peptides and proteins can be bound to ELISA plates. Inanother diagnostic method, the biological fluid is incubated on theantibody-bound plate and washed. Detection of any antigen-antibodycomplex, and qualitative measurement of the labeled MAb is performedconventionally, as described above.

Other useful assay formats include the filter cup and dipstick. In theformer assay, an antibody of this invention is fixed to a sintered glassfilter to the opening of a small cap. The biological fluid or sample (5mL) is worked through the filter. If Bin1 is present, it will bind tothe filter which is then visualized through a second Bin2 peptide orprotein. The dipstick assay involves fixing an antigen or antibody to afilter, which is then dipped in the biological fluid, dried and screenedwith a detector molecule.

Other diagnostic assays can employ the antigen(s) or fragments of thisinvention as nucleic acid probes or as anti-sense sequences, which canidentify the presence of infection in the biological fluid byhybridizing to complementary sequences produced by the pathogen in thebiological fluids. Such techniques, such as PCR, Northern or Southernhybridizations etc. are well known in the art.

It should be understood by one of skill in the art that any number ofconventional protein assay formats, particularly immunoassay formats, ornucleic acid assay formats, may be designed to utilize the isolatedantigens and antibodies or their nucleic acid sequences or anti-sensesequences of this invention for the detection of disorders associatedwith inappropriate/altered levels of functional Bin and/or formonitoring inappropriate levels of Bin1 in animals and humans. Thisinvention is thus not limited by the selection of the particular assayformat, and is believed to encompass assay formats which are known tothose of skill in the art.

VI. Diagnostic Kits

For convenience, reagents for ELISA or other assays according to thisinvention may be provided in the form of kits. Such kits are useful fordiagnosing conditions associated with dysfunctional Bin2 levels and/orBin1 levels, including cancers in a human or an animal sample. Such adiagnostic kit contains an antigen of this invention and/or at least oneantibody capable of binding an antigen of this invention, or the nucleicacid sequences encoding them, or their anti-sense sequences.

Alternatively, such kits may contain a simple mixture of such antigensor sequences, or means for preparing a simple mixture.

These kits can include microtiter plates to which the Bin2 peptides,proteins, antibodies, or nucleic acid sequences of the invention havebeen pre-adsorbed, various diluents and buffers, labeled conjugates forthe detection of specifically bound antigens or antibodies, or nucleicacids and other signal-generating reagents, such as enzyme substrates,cofactors and chromogens. Other components of these kits can easily bedetermined by one of skill in the art. Such components may includepolyclonal or monoclonal capture antibodies, antigen of this invention,or a cocktail of two or more of the antibodies, purified orsemi-purified extracts of these antigens as standards, MAb detectorantibodies, an anti-mouse or anti-human antibody with indicator moleculeconjugated thereto, an ELISA plate prepared for absorption, indicatorcharts for colorimetric comparisons, disposable gloves, decontaminationinstructions, applicator sticks or containers, and a sample preparatorcup. Such kits provide a convenient, efficient way for a clinicallaboratory to diagnose Bin2-associated conditions.

VII. Therapeutic Compositions and Methods

Compositions and methods useful for the treatment of conditionsassociated with inappropriate Bin2 levels are provided. Bin2 isexpressed predominantly in blood, e.g., hematopoictic, cells and isupregulated during monocytic differentiation. Thus, Bin2 levels havebeen detected in spleen and peripheral blood leukocytes, and in thymus,colon and placenta, and Bin2 RNA has been strongly expressed in severalhuman lymphoid and Lymphoid cell lines, including GM1500, ALL200, BVV173and HL60. Bin2 was induced during granulocytic differentiation of HL60cells, a promyelocytic leukemia cell line. Included among conditionsrelated to Bin2 expression include disorders associated with blood cellsand hepatocytes, including hepatocarcinomas and certain myeloid orlymphoid leukemias. Further, Bin2 may be useful in directing treatmentto disorders associated with inappropriate Bin1 functions, andparticularly, for inhibiting excessive Bin1 levels. TheseBin1-associated disorders include liver, colorectal, prostate, andbreast cancers, epithelial cell cancers, melanoma, and hyperplasticdisease states.

The therapeutic compositions of the invention may be formulated tocontain an anti-idiotype antibody of the invention, the Bin2 proteinitself or a fragment thereof, or nucleic acid sequences which directexpression of these antibodies, proteins or fragments thereof, includinganti-sense sequences. The therapeutic composition desirably contains0.01 μg to 10 mg protein. These compositions may contain apharmaceutically acceptable carrier, which facilitate administration ofthe compositions but are physiologically inert and/or nonharmful.Suitable carriers are well known to those of skill in the art andinclude, for example, saline. Alternatively, such compositions mayinclude conventional delivery systems into which protein of theinvention is incorporated. Optionally, these compositions may containother active ingredients, e.g., chemotherapeutics.

Carriers may be selected by one of skill in the art. Exemplary carriersinclude sterile saline, lactose, sucrose, calcium phosphate, gelatin,dextran, agar, pectin, peanut oil, olive oil, sesame oil, and water.Additionally, the carrier or diluent may include a time delay material,such as glycerol monostearate or glycerol distcarate alone or with awax. In addition, slow release polymer formulations can be used.

Optionally, this composition may also contain conventionalpharmaceutical ingredients, such as preservatives, or chemicalstabilizers. Suitable ingredients which may be used in a therapeuticcomposition in conjunction with the antibodies include, for example,casamino acids, sucrose, gelatin, phenol red, N-Z amine, monopotassiumdiphosphate, lactose, lactalbumin hydrolysate, and dried milk.

Alternatively, or in addition to the antibodies of the invention, otheragents useful in treating hepalocarcinoma or other conditions associatedwith dysfunctional Bin2 levels, are expected to be useful in reducing oreliminating disease symptoms.

Such agents may operate in concert with the therapeutic compositions ofthis invention. The development of therapeutic compositions containingthese agents is within the skill of one in the art in view of theteachings of this invention.

According to the method of the invention, a human or an animal may betreated for hepatocarcinoma by administering an effective amount of sucha therapeutic composition. An “effective amount” may be between about0.05 to about 1000 μg/mL of a Bin2 peptide, protein or antibody of theinvention. A suitable dosage may be about 1.0 mL of such an effectiveamount. Such a composition may be administered 1-3 times per day over a1 day to 12 week period. However, suitable dosage adjustments may bemade by the attending physician or veterinarian depending upon the age,sex, weight and general health of the human or animal patient.Preferably, such a composition is administered parenterally, preferablyintramuscularly or subcutaneously. However, it may also be formulated tobe administered by any other suitable route, including orally ortopically.

Still another composition contains Bin2 polynucleotide sequences whichcontain regulatory sequences which regulate expression of the proteinsof the invention in vivo or in vitro. Generally, a DNA-based compositioncontains 0.05 μg to 1 mg DNA per mL dose. Where the antigen-encodingpolynucleotide sequences are carried on a viral vector, a dose may be inthe range of 1×10⁻³ pfu to 1×10¹³ pfu per dose. However, the dose,timing and mode of administration of these compositions may bedetermined by one of skill in the art. Such factors as the age,condition, and the level of the Bin2 deficiency detected by thediagnostic methods described above, may be taken into account indetermining the dose, timing and mode of administration of thetherapeutic compositions of the invention. Generally, where treatment ofan existing cancer or hyperplastic state is indicated, a therapeuticcomposition of the invention is preferably administered in asite-directed manner and is repeated as needed. Such therapy may beadministered in conjunction with conventional therapies, includingradiation and/or chemotherapeutic treatments.

VIII. Drug Screening and Development

The Bin2 peptides, antibodies and polynucleotide sequences of thepresent invention are also useful in the screening and development ofchemical compounds or proteins which have utility as therapeutic drugsfor the treatment of cancers associated with inappropriate Bin2 levels.

Suitable assay methods may be readily determined by one of skill in theart. Where desired, and depending upon the assay selected, Bin2 may beimmobilized directly or indirectly (e.g., via an anti-Bin2 antibody) ona suitable surface, e.g., in an ELISA format. Such immobilizationsurfaces are well known. For example, a wettable inert bead may be used.Alternatively, Bin2 may be used in screening assays which do not requireimmobilization, e.g., in the screening of combinatorial libraries.Assays and techniques exist for the screening and development of drugscapable of binding to selected regions of Bin2. These include the use ofa phage display system for expressing the Bin2 proteins, and using aculture of transfected E. coli or other microorganism to produce theproteins for binding studies of potential binding compounds. See, forexample, the techniques described in G. Cesarini, FEBS Letters,307(1):66-70 (July 1992); H. Gram et al, J. Immunol. Meth., 161:169-176(1993); C. Summer et al., Proc. Natl. Acad. Sci. USA, 89:3756-3760 (May1992).

Other conventional drug screening techniques may be employed using theBin2 peptides, antibodies or polynucleotides of this invention. As oneexample, a method for identifying compounds which specifically bind to aBin2 protein can include simply the steps of contacting a selected Bin2protein with a test compound to permit binding of the test compound tothe Bin2 protein; and determining the amount of test compound, if any,which is bound to the Bin2 protein. Such a method may involve theincubation of the test compound and the Bin2 protein immobilized on asolid support.

Typically, a surface containing the immobilized ligand is permitted tocome into contact with a solution containing the Bin2 protein andbinding is measured using an appropriate detection system. Suitabledetection systems include, without limitation, the streptavidinhorseradish peroxidase conjugate and direct conjugation by a tag, e.g,fluorescein. Other systems are well known to those of skill in the art.This invention is not limited by the detection system used.

Another method of identifying compounds which specifically bind to aBin2 protein can include the steps of contacting a Bin2 proteinimmobilized on a solid support with both a test compound and the proteinsequence which is a receptor for Bin2 to permit binding of the receptorto the Bin2 protein; and determining the amount of the receptor which isbound to the Bin2 protein. The inhibition of binding of the normalprotein by the test compound thereby indicates binding of the testcompound to the Bin2 protein.

In still another method, interaction blockers may be identified. As anexample a simple plate assay could be used to screen for such blockers.In one embodiment of a plate assay one peptide/protein is bound to thedish and the other is added in an aqueous buffer (physiological KCl,e.g. 150 mM, and pH, eg. 7.5). Binding is monitored using an antibody tothe peptide which is conjugated to a fluorescent or enzymological marker(e.g., fluorescein or HRP). The primary antibody could also be detectedby using an anti-primary antibody that is so tagged. The bindingconditions are empirically optimized for salt, pH, metal, and detergentconditions. Under optimized binding conditions, the assay is run in sucha fashion that peptides or peptidomimetic drugs are added to the bindingbuffer. The positive control for blocking binding is provided byaddition of excess Bin protein, whereas the negative control is anunrelated protein or scrambled peptide. For example, a GST-Bin1 proteinis used to coat a plate and a Bin2 peptide used for binding. The peptideis directly identified by an antibody; alternatively, it is epitopetagged. Still alternately, the peptide itself is conjugated to themarker desired. This type of assay is amenable to high throughputscreening since it can be configured in a 96-well format.

Thus, through use of such methods, the present invention providescompounds capable of interacting with Bin2, or selected portionsthereof, and either enhancing or inhibiting its biological activity, asdesired. The assay methods described herein are also useful in screeningfor inhibition of Bin2. The solution containing the inhibitors may beobtained from any appropriate source, including, for example, extractsof supernatants from cultures of bioorganisms, extracts from organismscollected from natural sources, chemical compounds, and mixturesthereof.

The following examples illustrate the isolation and use of the Bin1sequences of the invention. These examples are illustrative only and donot limit the scope of the invention.

EXAMPLE 1 BIN2 is a Novel Bar Family Adaptor Protein that Lacks an SH3Domain

An anti-Bin1 polyclonal antisera was previously observed to crossreactwith Bin1-related polypeptides in cells [Sakamuro et al, Nature Genet.14: 69-77 (1996)), suggesting that additional uncharacterized members ofthe BAR family existed in mammalian cells. Subsequent epitope mapping ofthe main regions of Bin1 recognized by this antisera defined a majorepitope between the extreme C-terminus of the BAR domain [Wechsler-Reyaet al., Cancer Res. 57: 3258-3263 (1997)]. A TBLASTN search of theexpressed sequence tag (EST) database with the amino acid sequencecontaining this epitope identified a germinal B cell cDNA (Genbankaccession number AA452680) which encoded a polypeptide related to butnonidentical to amphiphysin or Bin1. Using this EST cDNA as a probe afull-length cDNA was obtained from a human leukocyte phage library andits complete DNA sequence was determined using standard methods [see,e.g., Sambrook et al, cited above]. A long open reading frame (ORF)identified in this cDNA encoded a 564 amino acid polypeptide rich inserine and glutamic acid with a predicted molecular weight of 61,709 Da(see FIGS. 1A-1C). Using the BLAST2 algorithm to compare Bin1 with thispolypeptide, termed Bin2 (Bridging INtegrator-2), the presence of acomplete BAR motif that had 61% identity and 75% similarity withboundaries of amino acid 1-249 of Bin2 and amino acid 1-251 of Bin1 (seeFIG. 2) was confirmed. In particular. Bin2 was identical to Bin1 withina region of the latter (amino acid 138-155) which is most highlyconserved region in BAR family proteins in evolution (G.C.P.,unpublished observations), whereas amphiphysin has a nonidenticalresidue in this region and was slightly less similar overall (data notshown). Bin2 lacked any canonical motifs other than the BAR domain (seeFIG. 3). In particular, it lacked an S1H3 domain which is found at theC-terminus of Bin1, amphiphysin, and RVS167. While RVS161 also lacks anSH3 domain, Bin2 differs in that it includes a large C-terminus thatextends beyond the BAR domain. Notably, Bin2 lacked sequences implicatedin normeuronal isoforms of Bin1 in interaction with c-Myc orTATA-binding protein in the nucleus (Sakamuro et al. cited above] or inalternately spliced neuronal isoforms in interaction with clathrin[Ramjaun and McPherson, J. Neurochem. 70: 2369-2376 (1998)] or AP-2 (P.de Camilli, pers. comm.). Thus, the structure of Bin2 suggested stronglythat it represented a nonredundant function. Southern analysis confirmedthe presence of Bin2 sequences in human genomic DNA (see below). It wasconcluded that Bin2 was a novel mammalian member of the BAR family ofadaptor proteins.

EXAMPLE 2 The Human BIN2 Gene is Located on Chromosome 4q221 andExhibits Aberrant Organization in Hepatoma Cells

The Bin2 cDNA was used to isolate three human Bin2 genomic BAC clones bystandard methods. Restriction analysis and Southern hybridization ofthese clones and comparison to Genomic Southern hybridizations confirmedthe presence of Bin2 sequences and ruled out the possibility that apseudoene was cloned. One of the clones, F727, was used to performfluorescence in situ hybridization (FISH) analysis of metaphasechromosomes isolated from normal peripheral blood lymphocytes,essentially as described in Cells: A Laboratory Manual, vol. 3, p.111.1-111.44, D. L. Spector et al, eds., Cold Spring Harbor Press, ColdSpring Harbor N.Y. 1998.

More particularly, fluorescence in situ hybridization (FISH) wasperformed using a Bin2 genomic BAC clone labeled by nick translationwith digoxigenin dUTP and metaphase chromosomes isolated fromPHA-stimulated normal peripheral blood lymphocytes. A biotin-labelledprobe specific for the centromere of chromosome 4 was prepared and alsoincluded in the hybridization. Slides were developed using afluoresceinated anti-digoxigenin antibodies and Texas red avidin.

Specific hybridization signals were detected on the long arm of a groupB chromosome consistent with chromosome 4 on the basis of size,morphology, and banding pattern. An experiment which included a secondprobe specific for the centromere sequences of chromosome 4 confirmedthis interpretation. A total of 80 metaphase cells analyzed with 73exhibiting specific labeling. Measurements of 10 specifically labeledchromosomes 4 demonstrated that the Bin2-specific hybridization signalwas located at a position 27% the distance from the centromere to thetelomere of chromosome arm 4q, an area corresponding to band 4q22.1. Twostudies have reported this region to be deleted in>50% of breastcarcinomas [Schwendel et al., Br. J. Cancer 78: 806-811 (1998)], with aneven higher frequency in tumors harboring BRCA1 mutations [Tirkkonen etal., Cancer Res. 57: 1222-1227 (1997)], and a third study has reportedthis region to be deleted in>75% of hepatocarinomas [Yeh et al,Gastroenterology 110: 184-192 (1996)]. The structure of the Bin2 gene innormal human foreskin, HepG2 or HLF hepatoma cells, and DU 145 prostatecarcinoma cells was compared by genomic Southern analysis to assess thepossibility of structural alteration.

For the Southern analysis, genomic DNA isolated from human foreskin,HepG2 or HLF hepatoma cells, or DU145 prostate carcinoma cells (as anegative control) was analyzed by standard Southern analysis using theBin2 cDNA as a hybridization probe. Both HepG2 and HLF exhibited normaland aberrant banding patterns relative to normal foreskin and DU145cells, which showed the same pattern as foreskin, consistent withalteration of at least one allele of the Bin2 gene in certain livertumors. Similar to DU145, genomic DNAs isolated from a panel ofnonmalignant and malignant B lymphoid cell lines (Daudi, Raji, 380,GM1500, G97, BVI 73) also did not exhibit aberrent organization of theBin2 gene. It was concluded that the Bin2 gene localized to chromosome4q22.1 within a region that was altered in hepatocellular tumors.

EXAMPLE 3 Evidence of Similarity Between BIN2, BIN1, and DAXX

In an effort to gain insight into the function of Bin2, the sequences inits central and C-terminal regions were compared to the DNA databaseusing the BLAST algorithm. Searches conducted with sequences derivedfrom the central region of Bin2, including a C-terminal part of the BARdomain, revealed a region (amino acid 179-336) that was similar to afunctionally undefined central region of Daxx, an adaptor proteinimplicated in Jun kinase (JNK) signaling and interactions with theprogrammed cell death receptor CD95/Fas in the cytosol and thecentromere-binding protein CENP-C in the nucleus [Chang et al, Science281: 1860-1863 (1998); Pluta et al. J. Cell Sci. 111:2029-2041 (1998);Yang et al. Cell 89: 1067-1076 (1997); Zhang et al., Nature 392: 296300(1998)]. The alignment included part of the BAR domain and Bin1 andamphiphysin could also be aligned with Daxx. Table I illustrates thesimilarity between central regions of Bin2 and Daxx. Amino acidsequences from the central region of Bin2 were used to query the DNAdatabase for related sequences using the TBLASTN algorithm. This figureprovides the selected hits and significance scores. The query sequencewas amino acid 179-336 in Bin) [SEQ ID NO: 2]. The score for the Daxxhit was more significant than the hit to the related yeast BAR proteinRVS161 (underlined). See, also, FIGS. 4A and 4B.

TABLE 1 Sequences producing significant alignments Score (bits) E Valuegi 1438562 gb U60884 MMU60884 117 1e-25 Mus musculus SH3P9 mRNA,complete . . . gi 550449 gb U07616 HSU07616 113 1e-24 Human amphiphysinmRNA, complete cds. gi 2199534 gb AF001383 AF001383 113 2e-24 Homosapiens amphiphysin II mRNA . . . gi 1710134 gb U68485 HSU68485 1117e-24 Human Box-dependent MYC-interacti . . . gi 2323471 gb AF015956AF015956 41 0.009 Homo sapiens Fas-binding protein . . . gi 976346 gbL32832 HUMZFHP 40 0.020 Homo sapiens zinc finger homeodomai . . . gi2745970 gb U84003 HSTSBIN5 39 0.058 Homo sapiens putative tumor suppr .. . gi 1122811 emb Z68217 CEF58G6 38 0.076 Caenorhabditis elegans cosmidF58 . . . gi 4417 emb X63315 SCRVS161 38 0.076 S. cerevisiae RVS 161gene gi 4057 emb X57185 SCNSR1 37 0.22 Yeast NSR1 gene for nuclearlocalizat . . . gi 29860 emb X55039 HSCENPB 36 0.29 Human hCENP-B genefor centromere a . . .

However, whereas the extent and significance of the alignments to Bin1and amphiphysin were relatively lower, the E value computed by BLAST forthe Bin2-Daxx alignment ranked it stronger than that the analogousalignment between Bin2 and the yeast BAR family member RVS 161 (9e-3versus 7.6e-2, respectively). The rank related in part to a sharedglutamate-rich segment found in Bin2 and Daxx but absent from other BARfamily members. It was concluded that Bin2 and Daxx shared a similarregion of structure within a central region of each polypeptide.

The database comparisons identified a region of similarity between thecentral domains of Bin2 and Daxx, a nucleocytosolic adaptor proteinlinked to cell survival decisions and chromosome function. Thefunctional implications of this relationship are unclear. Daxx wasinitially identified through its ability to interact with the deathdomain of CD95/Fas, a member of the tumor necrosis factor (TNF)superfamily of cell surface death receptors. Initial investigationssuggested a role for Daxx in death signaling by CD95/Fas [Yang et al.1997, cited above], but more recent studies have suggested that whileDaxx may have a role in JNK signaling by the receptor it is dispensablefor death signaling [Chang et al., Science 281: 1860-1863 (I 998); Changet al., Proc. Natl. Acad. Sci. USA 96: 1252-1256(1999)]. Another studyidentified Daxx through its ability to interact with CENP-C, acentromere-binding protein. While the meaning of this interaction is notyet known, consistent with some nuclear role Daxx has also been found tointeract with the cancer-related protein Pm1 and to localize to thesubnuclear domain ND10 where Pm1 can be found (G. Maul, pers. comm.).Interestingly, Pm1 and Bin1 have each been demonstrated to have a rolein certain types of programmed cell death [Quignonce at. Nat. Genet. 20:259-265 (1998); Wang et al., Nat. Genet. 20: 266-272 (1998)]. Therefore,it is tempting to speculate that the relationship between Daxx and Bin2may reflect some related connection to cell survival controls. Thispossibility would be consistent with the apparent role of RVS proteinsin yeast survival, following stresses which result from nutrientstarvation.

EXAMPLE 4 BIN2 is Expressed Predominantly in Hematopoietic Cells and isUpregulated During Monocytic Differentiation

Northern analyses of total RNAs isolated from human tissues and celllines were performed using the Bin2 cDNA as a hybridization probe toinvestigate the range of expression of Bin2 and to compare it withamphiphysin and Bin1 expression. Prior to Northern analysis, cells weretreated with DMSO or RA for 1, 3 or 5 days and RNA was isolated forNorthern analysis with Bin2 cDNA. The human tissues studied includedheart, brain, placenta, lung, liver, skeletal muscle, kidney, pancreas,spleen, thymus, prostate, testis, ovary, small intestine, colon, andperipheral blood leukocytes. The cell lines included: Raji, 380, 697,VM173, GM1500, ALL200, Daudi, HepG2, Caco-2, HCT116, LoVo, HBL100,ZR75-1, MCF7, BT20, SK-BR-3, A549, HLF, DU145, LNCaP, PC3, U373, U87-MG,and NCH2. Also determined was the level of Bin2 induction duringdifferentiation of HL60 promyelocytic leukemia cells to monocytes.

Northern hybridization of human tissue blots obtained from Clontechinitially suggested a broad range of expression, similar to Bin1 and incontrast to amphiphysin (results not shown). Two Bin2 messages werenoted in most tissues, suggesting alternate splicing as occurs in Bin1[Wechsler-Reya et al., J. Biol. Chem. 272: 31453-31458 (1997)]. Thehighest levels of Bin2 message were seen in spleen, peripheral bloodleukocytes, thymus, placenta, testis, colon, liver, and lung. Thepattern of expression more resembled Bin 1, which is ubiquitous butexpressed at highest levels in skeletal muscle, than amphiphysin, whichis essentially specific to the central nervous system. However,additional experiments suggested that Bin2 might be preferentiallyexpressed in hemapoietic cells and that the apparent pattern ofubiquitous expression reflected contamination of various tissues withhematopoietic cells, where Bin2 was expressed at high levels. Forexample, Bin2 message was barely detectable or undetectable by Northernanalysis in 21 human cell lines derived from a variety of tissues,including breast, lung, prostate, brain, connective tissue (fibroblast),liver, and colon, despite the detection of Bin2 in these tissues. Incontrast, Bin2 message was strongly expressed in several lymphoid celllines (GM1500, ALL200, BV173, Jurkat) and in myeloid cells (HL60) thatare derived from the hematopoictic lineage. Given that the Bin2 gene maybe altered in the cell lines examined, which were immortalized ormalignant, it was possible that the inability to detect expression wasdue to functional loss. The likelihood that Bin2 was predominantlyexpressed in hematopoietic cells was supported by “virtual” Northernanalyses, performed by comparing Bin2 sequences to the EST database,which provides information about the source of the cDNA. The majority ofidentical ESTs identified in this manner bad been cloned from cDNAlibraries derived from germinal B lymphoid cells, fetal liver (which isrich in B lymphoid cells), and placenta (which is rich in myeloidcells), although kidney libraries were also represented. One cell lineidentified to be positive for Bin2 expression was HL60 promyclocyticleukemia cells, which can be induced to differentiate to monocytes bytreatment with dimethyl sulfoxide (DMSO) or to granulocytes by treatmentwith retinoic acid (RA). To assess the possibility that Bin2 mayfunction in differentiated myeloid cells, it was determined whether thelevel of Bin2 message was altered during HL60 differentiation down themonocytic or granulocytic pathways. Northern analysis demonstrated thatBin2 levels were increased within 5 days of DMSO but not RA treatment.The level of Bin2 following induction was similar to that found in humanU937 myeloid cells and higher than in Jurkat T lymphoid cells. Theelevation of Bin2 during differentiation was reminiscent of a similarelevation of Bin1 which occurs during differentiation of skeletalmyoblasts and certain other cells including smooth muscle andkeratinocytes [Mao et al., Genomics 56: 51-58 (1999); Wechsler-Reya etal. Mol. Cell. Biol. 18: 566-575 (1998)]. It was concluded that Bin2 wasexpressed predominantly in hematopoietic cells and was likely tofunction in the myeloid lineage.

Bin2 is expressed predominantly in hemapoietic cells. This pattern ofexpression contrasts with amphiphysin, which is largely restricted tothe central nervous system, as well as with Bin1, which is highlyexpressed in muscle but otherwise ubiquitous. Induction of Bin2 messagewas documented in a model system for monocytic differentiation, insupport of the notion that Bin2 is likely to have an importantfunction(s) in the hematopoietic lineage. Whether the Bin2 gene hasimportant functions in other tissues is not yet clear. While expressionwas documented by Northern analysis in many normal tissues, theseresults could be ascribed to contamination of tissues with hemapoieticcells: while Bin2 message was abundant in lymphoid and myeloid cells, itwas undetectable in most benign and malignant non-hemapoietic cell linesexamined. It is conceivable that some of the deficits seen may be due tolosses in malignant settings, such as is the case with Bin1 [Sakamuro etal., Nature Genet. 14: 69-77 (1996)]. Consistent with this possibility,the Bin2 gene is located at chromosome 4q22.1, a region frequentlydisrupted in hepatocarcinoma, and aberrant organization of the gene wasobserved in two liver tumor lines. Bin2 was not expressed in these linesbut little effect of ectopic Bin2 on the proliferation of these celllines in vitro was noted, so the significance of the aberrant geneorganization and possible loss of expression remains unclear.

EXAMPLE 5 BIN2 and BIN1 Form a Stable Complex That Requires the BarDomain

Stable complexes have been reported in yeast between RVS161 and RVS167and in brain between amphiphysin and brain-specific splice isoforms ofBin1 [Navarro et al., Biochim. Biophys. Acta. 1343: 187-192 (1997);Wigge et al., Mol. Biol. Cell 8: 2003-2015 (1997)]. Since Bin1 isexpressed in hematopoietic cells, whether Bin2 and Bin1 could also forma stable biochemical complex was investigated. For communoprecipitationexperiments, Bin1 was in vitro translated in the presence or absence ofBin2 and complex formation was assessed by SDS-PAGE and fluorographyafter immunoprecipitation with the Bin1 monoclonal antibody 99D[Wechsler-Reya et al., Cancer Res. 57: 3258-3263 (1997)].

Bin2 and Bin1 were in vitro translated with empty vector or cotranslatedwith each other in the presence of ³⁵S-methionine. Products werefractionated on SDS-PAGE gels and fluorographed or subjected toimmunoprecipitation with the anti-Bin1 antibody 99D [Wechsler-Reya etal., cited above] before fractionation. Bin2 migrated with an apparentMW 82 kD, which is greater than the predicted MW ˜61 kD, reminiscent ofa similar aberrent mobility displayed by Bin1, which has an apparent MW˜68 kD and a predicted MW ˜50 kD. Bin2 was not immunoprecipitated by 99Dunless cotranslated with Bin1.

Using a set of deletion mutants (Δ124-207, Δ1-122, Δ152-207, Δ143-148)the BAR domain in Bin1 was demonstrated to be required for Bin2interaction. Deletion of the C-terminal region of BAR decreased bindingefficiency, whereas deletion of residues 143-148 within a loop regionimplicated in effector signaling and representing the most highlyconserved part of the BAR domain in Bin1 bad no effect. Deletion of theN-terminal region of BAR (residues 1-122) eliminated binding completely,arguing that the N-terminus of Bin1 was crucial for interaction withBin2. Other deletion mutants of Bin1 lacking the so-called Uniquecentral region, the Myc-binding domain, or the SH3 domain, interactedwith Bin2 with the same efficiency as wild-type Bin1 (data not shown).It was concluded that Bin1 and Bin2 formed a BAR domain-dependentcomplex in cells.

Bin1 and Bin2 were shown to form a stable biochemical complex, in themanner of RVS161 and RVS167 in yeast or amphiphysin and neuronal spliceisoforms of Bin1 in mammalian cells [Navarro et al., cited above (1997);Wigge et al., cited above (1997)], and the association depended upon theintegrity of the BAR domain. Bin2 did not affect the tumor suppressorproperties of Bin1 that are manifested in HepG2 cells [Sakamuro et al.,Nature Genet. 14: 69-77 (1996)]. This may reflect different requirementsfor each activity, since Bin2 association rested on an N-terminal BARdeterminant whereas the tumor suppressor activity of Bin1 rests upon aC-terminal BAR determinant. Evidence that BAR domains encode uniqueactivities and are not functionally equivalent is provided by domainswapping studies performed in yeast [Sivadon et al., FEBS Lett. 417:21-27 (1997)]. Thus, the BAR domain of Bin2 may have unique features,perhaps related to Bin1 regulation rather than effector signaling. Infuture work, it will be important to determine the physiologicalfunctions of Bin2 and how they are manifested independently or in anintegrated manner with the functions of Bin1.

EXAMPLE 6 BIN2 Lacks In Vitro Growth Inhibitory Properties

Bin1 has tumor suppressor properties in certain malignant cells[Sakamuro et al. 1996, cited above], so whether Bin2 had any similareffects and/or whether it could influence the growth inhibitory activityof Bin1 was investigated. HepG2 hepatoma cells lack endogenous Bin1 andBin2 expression so they provided a useful background to perform theseexperiments. Another cell line which lacks Bin2 expression, A549 lungcarcinoma cells, was also used in these experiments. Cells weretransfected with the same expression vector used above, which carries aneomycin resistance cassette, and stably transformed cells were selectedin growth media containing G418.

A ˜2-fold decrease in colony formation efficiency relative to emptyvector was noted in HepG2 cells but not in A549 cells (see FIG. 5A).These observations suggested that Bin2 may be weakly growth inhibitory.To confirm this, HepG2 colonies were ring-cloned, expanded into celllines, and examined for ectopic Bin2 expression by Northern analysis.For Northern analysis, total cytoplasmic RNA was isolated from coloniesthat were ring-cloned and expanded into cell lines. Northern analysiswas performed using Bin2 cDNA as probe. Robust levels of Bin2 mRNA weredetected in several independent cell lines which did not exhibit anysigns of growth inhibition, confirming that accumulation of Bin messagewas compatible with HcpG2 proliferation To determine if Bin2 couldrelieve or augment growth suppression by Bin1, a similar set of colonyformation experiments was performed in HepG2 except that untagged emptyvector or Bin2 vector was cotransfected with a neomycin resistancegene-tagged Bin1 vector [Sakamuro et al. 1996, cited above]. The numberof colonies which emerged using Bin2 vectors was similar to thoseproduced with control vector, indicating that Bin2 did not affect theability of Bin1 to suppress HepG2 cell growth (see FIG. 5B). It wasconcluded that Bin2 lacked the strong growth inhibitory properties inHep G2 cells that are inherent to Bin1.

All above-noted references and priority document are incorporated hereinby reference. Numerous modifications and variations of the presentinvention are included in the above-identified specification and areexpected to be obvious to one of skill in the art. Such modificationsand alterations to the compositions and processes of the presentinvention are believed to be encompassed in the scope of the claimsappended hereto.

4 1 2196 DNA Homo sapiens CDS (28)..(1719) 1 gcggccgcgt cgacgggagttggcagg atg gca gag ggc aag gca ggc ggc gcg 54 Met Ala Glu Gly Lys AlaGly Gly Ala 1 5 gcc ggc ctc ttc gcc aag cag gtg cag aag aag ttt agc agggcc cag 102 Ala Gly Leu Phe Ala Lys Gln Val Gln Lys Lys Phe Ser Arg AlaGln 10 15 20 25 gag aag gtg ctg cag aaa ttg ggg aaa gct gta gaa acc aaagat gaa 150 Glu Lys Val Leu Gln Lys Leu Gly Lys Ala Val Glu Thr Lys AspGlu 30 35 40 cga ttt gaa caa agc gct agc aac ttc tac caa caa cag gca gaaggc 198 Arg Phe Glu Gln Ser Ala Ser Asn Phe Tyr Gln Gln Gln Ala Glu Gly45 50 55 cac aag ctg tac aag gac ctg aag aac ttc ctt agt gca gtc aaa gtg246 His Lys Leu Tyr Lys Asp Leu Lys Asn Phe Leu Ser Ala Val Lys Val 6065 70 atg cat gaa agt tca aaa aga gtg tca gaa acc ctg cag gag atc tac294 Met His Glu Ser Ser Lys Arg Val Ser Glu Thr Leu Gln Glu Ile Tyr 7580 85 agc agc gag tgg gac ggt cat gag gag ctg aag gcc atc gta tgg aat342 Ser Ser Glu Trp Asp Gly His Glu Glu Leu Lys Ala Ile Val Trp Asn 9095 100 105 aat gat ctc ctt tgg gaa gac tac gag gag aaa ctg gct gac caggct 390 Asn Asp Leu Leu Trp Glu Asp Tyr Glu Glu Lys Leu Ala Asp Gln Ala110 115 120 gta agg acc atg gaa atc tat gtt gcc cag ttc agt gaa att aaggag 438 Val Arg Thr Met Glu Ile Tyr Val Ala Gln Phe Ser Glu Ile Lys Glu125 130 135 aga att gcc aag cgg ggt cgg aaa ctc gtg gac tat gac agt gcccga 486 Arg Ile Ala Lys Arg Gly Arg Lys Leu Val Asp Tyr Asp Ser Ala Arg140 145 150 cac cac ctg gag gca gtg cag aat gcc aag aaa gat gag gcc aagact 534 His His Leu Glu Ala Val Gln Asn Ala Lys Lys Asp Glu Ala Lys Thr155 160 165 gcc aag gca gag gaa gag ttc aac aaa gcc cag act gtg ttt gaagat 582 Ala Lys Ala Glu Glu Glu Phe Asn Lys Ala Gln Thr Val Phe Glu Asp170 175 180 185 ctg aac caa gaa cta cta gag gag ctg cct att ctt tat aatagt cgt 630 Leu Asn Gln Glu Leu Leu Glu Glu Leu Pro Ile Leu Tyr Asn SerArg 190 195 200 att ggc tgc tat gtg acc atc ttc caa aac att tcc aac ttgagg gat 678 Ile Gly Cys Tyr Val Thr Ile Phe Gln Asn Ile Ser Asn Leu ArgAsp 205 210 215 gtc ttc tac agg gaa atg agc aag ctg aac cac aat ctc tacgag gtg 726 Val Phe Tyr Arg Glu Met Ser Lys Leu Asn His Asn Leu Tyr GluVal 220 225 230 atg agc aaa ctg gag aag caa cat tcc aat aaa gtc ttt gtggtg aag 774 Met Ser Lys Leu Glu Lys Gln His Ser Asn Lys Val Phe Val ValLys 235 240 245 gga ctg tca agc agc agc agg cgc tct tta gtc att tct ccccca gtt 822 Gly Leu Ser Ser Ser Ser Arg Arg Ser Leu Val Ile Ser Pro ProVal 250 255 260 265 cga aca gct aca gtc tcc agt cct ctt acc tca cct actagt ccc tct 870 Arg Thr Ala Thr Val Ser Ser Pro Leu Thr Ser Pro Thr SerPro Ser 270 275 280 aca ctt tcc ttg aag agt gag agt gaa tct gtc tca gcaact gaa gat 918 Thr Leu Ser Leu Lys Ser Glu Ser Glu Ser Val Ser Ala ThrGlu Asp 285 290 295 ctg gca cct gat gca gcc caa ggg gaa gac aat tct gagatc aag gag 966 Leu Ala Pro Asp Ala Ala Gln Gly Glu Asp Asn Ser Glu IleLys Glu 300 305 310 ctc tta gaa gag gag gaa ata gag aag gaa gga tct gaagca agc tcc 1014 Leu Leu Glu Glu Glu Glu Ile Glu Lys Glu Gly Ser Glu AlaSer Ser 315 320 325 tct gag gaa gat gac cct cta cca gcc tgc aat ggc cccgcc cag gcc 1062 Ser Glu Glu Asp Asp Pro Leu Pro Ala Cys Asn Gly Pro AlaGln Ala 330 335 340 345 cag ccc tct cct acc act gag agg gcc aag tcc caggag gaa gtt ctc 1110 Gln Pro Ser Pro Thr Thr Glu Arg Ala Lys Ser Gln GluGlu Val Leu 350 355 360 ccc agc tcc aca act cca tca cca ggc gga gcc ctgagc cct tca ggg 1158 Pro Ser Ser Thr Thr Pro Ser Pro Gly Gly Ala Leu SerPro Ser Gly 365 370 375 cag cct tca tca tct gcc aca gaa gta gtc ctc cgaacc cgc acc gca 1206 Gln Pro Ser Ser Ser Ala Thr Glu Val Val Leu Arg ThrArg Thr Ala 380 385 390 agt gaa gga tct gaa caa cca aag aag aga gcc tctatc cag agg acc 1254 Ser Glu Gly Ser Glu Gln Pro Lys Lys Arg Ala Ser IleGln Arg Thr 395 400 405 tca gca ccc cct agt agg cct cct cca ccc aga gccact gca agc ccc 1302 Ser Ala Pro Pro Ser Arg Pro Pro Pro Pro Arg Ala ThrAla Ser Pro 410 415 420 425 agg ccc tcc tca ggg aac ata cct tcc agc cctaca gcc tct gga ggg 1350 Arg Pro Ser Ser Gly Asn Ile Pro Ser Ser Pro ThrAla Ser Gly Gly 430 435 440 ggt tca ccc acc agc cct agg gcc tcc ttg gggact ggg act gca agt 1398 Gly Ser Pro Thr Ser Pro Arg Ala Ser Leu Gly ThrGly Thr Ala Ser 445 450 455 cct agg acc tcc cta gag gtc tct cct aat ccagaa cca cca gag aag 1446 Pro Arg Thr Ser Leu Glu Val Ser Pro Asn Pro GluPro Pro Glu Lys 460 465 470 cca gta aga act cct gag gcc aaa gaa aat gaaaac atc cac aat cag 1494 Pro Val Arg Thr Pro Glu Ala Lys Glu Asn Glu AsnIle His Asn Gln 475 480 485 aac cct gaa gaa ctt tgt act tcc ccc acc ttaatg aca tct cag gtt 1542 Asn Pro Glu Glu Leu Cys Thr Ser Pro Thr Leu MetThr Ser Gln Val 490 495 500 505 gct tca gag cct gga gag gca aag aag atggaa gac aag gaa aag gat 1590 Ala Ser Glu Pro Gly Glu Ala Lys Lys Met GluAsp Lys Glu Lys Asp 510 515 520 aat aag ctt atc tca gct gac tcc tcg gagggc caa gac cag ctt caa 1638 Asn Lys Leu Ile Ser Ala Asp Ser Ser Glu GlyGln Asp Gln Leu Gln 525 530 535 gtc tcc atg gta cca gaa aac aac aac ctcaca gca cct gaa cct caa 1686 Val Ser Met Val Pro Glu Asn Asn Asn Leu ThrAla Pro Glu Pro Gln 540 545 550 gaa gag gta tcc aca agt gaa aat cca caactc tgaagagaaa ctaccaagac 1739 Glu Glu Val Ser Thr Ser Glu Asn Pro GlnLeu 555 560 tcctcctgcc ccaaacctcg ccagagaagc tcttcaacca gagggtataggtcagaggga 1799 tataagagcc agcatccatc cctgggttct cagtaggaat gctggtgctgtctaaagacc 1859 tggcattaat ggaggcggag gagcagcctt acgggaggga tggagggaggcaggctgggg 1919 agaagagaac attagactca gggaatattt aattctggtt ttagcattattagaataaga 1979 ctttatacat taactaaagt ggagctttaa tcactataaa aagcaaaagtatntatagac 2039 acagacactt gcctatacag agacataacc acacacactc agaggatagtgaacaaatct 2099 gtctttgact tacgacccat tttgcaagac ttaaagccga aagaacacattttcagattg 2159 ttaaataaag tctgattctg actaaaaaaa aaaaaaa 2196 2 564 PRTHomo sapiens 2 Met Ala Glu Gly Lys Ala Gly Gly Ala Ala Gly Leu Phe AlaLys Gln 1 5 10 15 Val Gln Lys Lys Phe Ser Arg Ala Gln Glu Lys Val LeuGln Lys Leu 20 25 30 Gly Lys Ala Val Glu Thr Lys Asp Glu Arg Phe Glu GlnSer Ala Ser 35 40 45 Asn Phe Tyr Gln Gln Gln Ala Glu Gly His Lys Leu TyrLys Asp Leu 50 55 60 Lys Asn Phe Leu Ser Ala Val Lys Val Met His Glu SerSer Lys Arg 65 70 75 80 Val Ser Glu Thr Leu Gln Glu Ile Tyr Ser Ser GluTrp Asp Gly His 85 90 95 Glu Glu Leu Lys Ala Ile Val Trp Asn Asn Asp LeuLeu Trp Glu Asp 100 105 110 Tyr Glu Glu Lys Leu Ala Asp Gln Ala Val ArgThr Met Glu Ile Tyr 115 120 125 Val Ala Gln Phe Ser Glu Ile Lys Glu ArgIle Ala Lys Arg Gly Arg 130 135 140 Lys Leu Val Asp Tyr Asp Ser Ala ArgHis His Leu Glu Ala Val Gln 145 150 155 160 Asn Ala Lys Lys Asp Glu AlaLys Thr Ala Lys Ala Glu Glu Glu Phe 165 170 175 Asn Lys Ala Gln Thr ValPhe Glu Asp Leu Asn Gln Glu Leu Leu Glu 180 185 190 Glu Leu Pro Ile LeuTyr Asn Ser Arg Ile Gly Cys Tyr Val Thr Ile 195 200 205 Phe Gln Asn IleSer Asn Leu Arg Asp Val Phe Tyr Arg Glu Met Ser 210 215 220 Lys Leu AsnHis Asn Leu Tyr Glu Val Met Ser Lys Leu Glu Lys Gln 225 230 235 240 HisSer Asn Lys Val Phe Val Val Lys Gly Leu Ser Ser Ser Ser Arg 245 250 255Arg Ser Leu Val Ile Ser Pro Pro Val Arg Thr Ala Thr Val Ser Ser 260 265270 Pro Leu Thr Ser Pro Thr Ser Pro Ser Thr Leu Ser Leu Lys Ser Glu 275280 285 Ser Glu Ser Val Ser Ala Thr Glu Asp Leu Ala Pro Asp Ala Ala Gln290 295 300 Gly Glu Asp Asn Ser Glu Ile Lys Glu Leu Leu Glu Glu Glu GluIle 305 310 315 320 Glu Lys Glu Gly Ser Glu Ala Ser Ser Ser Glu Glu AspAsp Pro Leu 325 330 335 Pro Ala Cys Asn Gly Pro Ala Gln Ala Gln Pro SerPro Thr Thr Glu 340 345 350 Arg Ala Lys Ser Gln Glu Glu Val Leu Pro SerSer Thr Thr Pro Ser 355 360 365 Pro Gly Gly Ala Leu Ser Pro Ser Gly GlnPro Ser Ser Ser Ala Thr 370 375 380 Glu Val Val Leu Arg Thr Arg Thr AlaSer Glu Gly Ser Glu Gln Pro 385 390 395 400 Lys Lys Arg Ala Ser Ile GlnArg Thr Ser Ala Pro Pro Ser Arg Pro 405 410 415 Pro Pro Pro Arg Ala ThrAla Ser Pro Arg Pro Ser Ser Gly Asn Ile 420 425 430 Pro Ser Ser Pro ThrAla Ser Gly Gly Gly Ser Pro Thr Ser Pro Arg 435 440 445 Ala Ser Leu GlyThr Gly Thr Ala Ser Pro Arg Thr Ser Leu Glu Val 450 455 460 Ser Pro AsnPro Glu Pro Pro Glu Lys Pro Val Arg Thr Pro Glu Ala 465 470 475 480 LysGlu Asn Glu Asn Ile His Asn Gln Asn Pro Glu Glu Leu Cys Thr 485 490 495Ser Pro Thr Leu Met Thr Ser Gln Val Ala Ser Glu Pro Gly Glu Ala 500 505510 Lys Lys Met Glu Asp Lys Glu Lys Asp Asn Lys Leu Ile Ser Ala Asp 515520 525 Ser Ser Glu Gly Gln Asp Gln Leu Gln Val Ser Met Val Pro Glu Asn530 535 540 Asn Asn Leu Thr Ala Pro Glu Pro Gln Glu Glu Val Ser Thr SerGlu 545 550 555 560 Asn Pro Gln Leu 3 252 PRT Homo sapiens 3 Met Ala GluMet Gly Ser Lys Gly Val Thr Ala Gly Lys Ile Ala Ser 1 5 10 15 Asn ValGln Lys Lys Leu Thr Arg Ala Gln Glu Lys Val Leu Gln Lys 20 25 30 Leu GlyLys Ala Asp Glu Thr Lys Asp Glu Gln Phe Glu Gln Cys Val 35 40 45 Gln AsnPhe Asn Lys Gln Leu Thr Glu Gly Thr Arg Leu Gln Lys Asp 50 55 60 Leu ArgThr Tyr Leu Ala Ser Val Lys Ala Met His Glu Ala Ser Lys 65 70 75 80 LysLeu Asn Glu Cys Leu Gln Glu Val Tyr Glu Pro Asp Trp Pro Gly 85 90 95 ArgAsp Glu Ala Asn Lys Ile Ala Glu Asn Asn Asp Leu Leu Trp Met 100 105 110Asp Tyr His Gln Lys Leu Val Asp Gln Ala Leu Leu Thr Met Asp Thr 115 120125 Tyr Leu Gly Gln Phe Pro Asp Ile Lys Ser Arg Ile Ala Lys Arg Gly 130135 140 Arg Lys Leu Val Asp Tyr Asp Ser Ala Arg His His Tyr Glu Ser Leu145 150 155 160 Gln Thr Ala Lys Lys Lys Asp Glu Ala Lys Ile Ala Lys AlaGlu Glu 165 170 175 Glu Leu Ile Lys Ala Gln Lys Val Phe Glu Glu Met AsnVal Asp Leu 180 185 190 Gln Glu Glu Leu Pro Ser Leu Trp Asn Ser Arg ValGly Phe Tyr Val 195 200 205 Asn Thr Phe Gln Ser Ile Ala Gly Leu Glu GluAsn Phe His Lys Glu 210 215 220 Met Ser Lys Leu Asn Gln Asn Leu Asn AspVal Leu Val Gly Leu Glu 225 230 235 240 Lys Gln His Gly Ser Asn Thr PheThr Val Lys Ala 245 250 4 87 PRT Homo sapiens 4 Ala Gln Asp Ala Phe ArgAsp Val Gly Ile Arg Leu Gln Glu Arg Arg 1 5 10 15 His Leu Asp Leu IleTyr Asn Phe Gly Cys His Leu Thr Asp Asp Tyr 20 25 30 Arg Pro Gly Val AspPro Ala Leu Ser Tyr Pro Val Ser Ala Arg Arg 35 40 45 Leu Arg Glu Asn ArgIle Leu Ala Leu Ser Arg Leu Asp Gln Val Ile 50 55 60 Ser Phe Tyr Ala MetLeu Gln Asp Gly Gly Glu Glu Gly Lys Lys Lys 65 70 75 80 Lys Arg Arg AlaArg Leu Asp 85

What is claimed is:
 1. An isolated Bridging Integrator-2 (Bin2) proteinhaving the amino acid sequence of SEQ ID NO:
 2. 2. The Bin2 peptide orprotein according to claim 1 selected from the group consisting of: (a)amino acid sequence of SEQ ID NO: 2 with conservative amino acidsubstitution thereof; and (b) a fusion protein comprising the amino acidsequence of (a) or (b) and a fusion partner; and (c) a fusion proteincomprising the amino acid sequence of SEQ ID NO: 2 and a fusion partner.3. An isolated Bridging Integrator-2 (Bin21 peptide having the sequenceof amino acids 1 to 221 of SEQ ID NO:
 2. 4. The Bin2 protein accordingto claim 2, wherein the fusion partner is selected from the groupconsisting of glutationine-S-transferase, β-galactosidase,poly-histidine and maltose binding protein.
 5. A composition comprisingthe Bin2 protein of claim 1 or claim 2 and a pharmaceutically acceptablecarrier.